Cystic fibrosis (CF), a single gene defect located at 31q of human chromosome 7, affects 1:2,500 births and leads to a premature respiratory death. Although several human CF gene trials are underway using adenoviral CFTR constructs, major questions regarding the basic biology of infection of CFTR or other genes into the airway epithelium remain. Is over- expression of CFTR safe in epithelial cells of human? Do infected epithelial cells differentiate normally? Are all airway sub-populations infected equally? Are all airway sub-populations transduced equally? Are airway stem cells infected but not transduced until they develop a differentiated phenotype? Is mucin production and function normal? Do viral vectors induce pro inflammatory cytokines? The purpose of this proposal is to determine which airway epithelial cell compartments can be infected and transduced and to determine whether over-expression of CFTR alters airway epithelial cell differentiation and function. We hypothesize that infection will occur more efficiently in the basal and secretory populations, and that the rheological properties of mucin will be altered with increased expression of CFTR. Sub=populations of rabbit epithelial cells will be infected with a retroviral and adeno-associated viral vectors, initially with a marker genes and later with a CFTR construct. Two airway epithelial cell culture methods will be used: monolayers to measure infection and transduction efficiency, ion fluxes, mucin production and chemokine up regulation and organotypic cultures to determine airway epithelial cell differentiation. Once safety and efficacy are established in vitro, rabbits will be infected in vivo a=with AAV marker genes and with AAV-CFTR construct to determine toxicity and duration of CFTR expression. We hypothesize that there will be a low infection rate with AAV vectors, but there will be long term expression as there is genomic integration. These experiments will help to establish an essential foundation for the production of effective and safe vectors for human CF therapy.